There are many procedures performed that involve the manipulation of small particles such as cells, bacteria, yeast, fungi, viruses, protozoa, sperm, eggs, embryos, larvae, pollen, beads, ink particles and the like. Generally, the manipulation of small particles is inherently difficult because the particles are too small to be visualised with the naked eye.
Where a procedure is performed that involves adding small particles to a vessel (for example, a test tube) there is generally no simple technology available which allows one to know exactly, or at least with a minimal degree of error, how many particles have been added. Typically, a suspension of the particles would be prepared and then the suspension analysed (for example, enumeration by microscopy or culture on an agar plate) to estimate the number of particles per volume of liquid. An aliquot of this suspension, containing an estimated number of particles, would then be used without the exact number of particles in the aliquot being known.
There are various devices available that can accurately dispense a small volume of fluid. Pipettes are routinely used for this purpose and are able to accurately and reliably dispense volumes ranging from 0.0001 ml up to 20 ml. When using a pipette to manipulate small particles such as cells, the actual number of cells within the volume of fluid that is being dispensed is unknown and can not be controlled. Only the volume of fluid can be controlled.
Devices such as pipettes work very well for dispensing fluids that contain chemicals or soluble molecules in solution. This is because the chemicals or soluble molecules are evenly dispersed throughout the fluid (ie dissolved). This is not the case with particles such as cells. Particles are typically randomly distributed throughout the fluid. This means that two identical volumes of fluid taken from a suspension of particles will not contain the same number of particles. Devices such as pipettes are therefore not able to dispense known numbers of particles with a high degree of accuracy and precision. There is therefore a need for methods that can be used to accurately dispense known numbers of particles such as cells or microorganisms.
Flow cytometry is a technique that can be used to dispense particles. Flow cytometry can be used to physically sort particles such as cells using information from the various detectors as discriminators. Sorting enables purification of a particular particle type from a mixture. Flow cytometers, however, typically sort particles one at a time.
The standard method of sorting particles by flow cytometry is known as droplet deflection sorting. It relies on the use of a piezoelectric transducer in the flow cell to create droplets of sheath fluid. An alternating electrical current is passed across the transducer causing the flow cell to vibrate up and down at the same frequency as the current. The vibration of the flow cell causes undulations to form in the sheath fluid once it has left the flow cell. Further downstream from the flow cell the undulations in the stream of sheath fluid become more and more defined until the stream breaks up into droplets. The last undulation in the stream before the stream breaks up into droplets is known as the last attached droplet.
If a particle is to be collected, then an electric charge is placed on the sheath fluid at the exact time the particle is in the last attached droplet. The charge occurs for the duration of one vibration of the piezoelectric crystal. This results in a single droplet, the one containing the particle to be sorted, being charged. Further downstream from the flow cell the stream of droplets passes between two plates, one positively and one negatively charged. As the charged droplet passes between the plates it is diverted from the main stream of droplets enabling it to be collected. This sorting process can be performed at a rate of several thousands times per second using a modern cytometer.
Flow cytometers that use droplet deflection sorting, however, are expensive, large sophisticated instruments that require at least daily alignment by a highly skilled operator. Setting up the sorting is also difficult and requires a number of calibrations including calculation of the length of time that it takes a particle to travel from the interaction region to the last attached droplet. This length of time is known as the droplet delay. Once the sorting has been set up it has to be monitored closely to ensure that the droplet delay does not change.
A limitation of droplet deflection sorting is that it is not able to create droplets that contain more than one particle. A further problem with droplet deflection sorting is that it can create aerosols and is therefore not suitable for the analysis of biologically harsh conditions that are not particularly suitable for retaining viability or integrity of living cells. The difficulties with the use of droplet delay sorting have restricted the use of the technology to specialised research laboratories.
An alternative form of flow cytometry sorting is described in the U.S. Pat. No. 5,030,002 “Method and apparatus for sorting particles with a moving capture tube”. This sorting process uses a capture tube that is mechanically moved in and out of the sample stream to capture a particle. This allows the system to sort particles by detecting certain properties of the particles and then directing the particles one at a time either into the container or into the waste container. For example, this sorting process is utilised by the Becton Dickinson FACScalibur flow cytometer. This flow cytometer is simple to operate and requires no calibration or complex set-up procedure to be performed prior to sorting particles. It is simply a case of switching the instrument on, analysing a sample and sorting the particles of interest.
A limitation of this type of sorting flow cytometer, however, is the speed at which particles can be sorted. The capture tube can typically move in and out of the stream of particles at a speed of 300 times per second. The system is designed to sort one particle at a time. Thus 300 is the maximum number of particles that can be sorted per second with such prior art cytometers.
The present applicant has been able to produce standards having accurate very small numbers (less than 1000 and typically around 100 or less) of microorganisms (U.S. Pat. No. 6,780,581; WO 2003/02095). Unfortunately, the process used to make these standards cannot produce standards having greater that about 1000 microorganisms. Although there is a long felt need for standards having accurate numbers of particles up to around 1,000,000 such standards cannot be produced on a reasonable commercial scale. As a result of the limitations of existing metering devices, it has not been possible to produce accurate relatively high numbers of particles such as cells or microorganisms for use as standards or controls.
The present inventors have now developed methods to produce accurate and reproducible standards having up from 1000 to 1,000,000 particles.